Twenty-one
plants were identified through preliminary field surveys and seven were selected
for in vitro anti-helminthic activity against Ascaris suum. Of the seven
plants that were initially screened, five gave appreciable positive results
while two did not. The ED50s obtained were: 1.62mg/ml (Tetradenia
riparia), 4.13mg/ml (Cassia occidentalis), 12.50 mg/ml (Carica
papaya) and 16.75mg/ml (Momordica foetida). The research findings showed
that Tetradenia riparia, Cassia occidentalis, Carica papaya, Momordica foetida and Erythrina abysinnica may be of value in the
treatment of helminthiasis; whereas Moringa oleifera and Cannabis
sativa are probably ineffective or of limited value for the same purpose.

Key words: Ascaricidal, Uganda, Medicinal Plants

Introduction

Helminths are of
major public health and economic importance to both man and livestock
throughout the tropics. It is estimated that 60-80% of the worlds population
is affected by helminths with a vast majority of these in developing countries
(Fansworth, 1988). In man, diseases such as schistosomiasis, ascariasis and ancylostomiasis
cause untold suffering to millions of people worldwide, especially in
Sub-Saharan Africa. The effects of such infestations include gastroenteritis,
anemia, stunted growth, blindness and lameness among others. In the livestock
industry, similar conditions are seen that greatly affect productivity and
hence economic output of the livestock sector. Here, helminths cause a
multitude of problems such as poor weight gain, diarrhea, respiratory problems
and even mortalities in severe cases (Schmidt and Roberts, 1985). Ascariasis,
for instance, affects about one quarter of the worlds population, especially
the growth and nutritional status of children (Latham, 1977).

Control of
helminthiasis has therefore been the centre of focus in biomedical research
since time immemorial. Both the medical and veterinary professions have tried
to control helminthiasis by
administration of synthetic drugs (Ssebuguzi, 2000). However, these drugs are
becoming increasingly expensive with some having serious side effects (Siddiqui
and Hussein, 1992).

Despite the
large number of drugs and medicines available for treatment of all forms of
diseases, the demand for herbal medicines has steadily increased over the past
decade worldwide. However, a great majority of them are not assessed for their
quality, safety or licensed as medicines (Alte, 1993). Little is known or
documented about the usefulness, effectiveness or potential of such medicines.
With the onset of modernization of agriculture and other western influences,
such knowledge is greatly threatened and could totally be lost with the passing
generations. It is prudent therefore to urgently research more on this field
and generate vital data that could be necessary to revitalize and preserve such
knowledge. Previous studies in our laboratories have demonstrated antibacterial
and antifungal (Olila et al. 2001a) as well as antiviral and trypanocidal (Olila
et al., 2001b; Odyek et al., 1993; Olila et al., 2002) activity in
Ugandan medicinal plants.

Here we report
findings from studies designed to screen local plants that are used in the
treatment of helminthiasis in livestock in some parts of Uganda, where human
and veterinary services are still very poor; being compounded by many people
living in rural areas several kilometers from health centers. This has resulted
in a large proportion of the population relying on traditional methods of
treatment, using herbal extracts, which have been claimed to produce beneficial
responses. These remedies are not only more readily available and acceptable
but could also be cheaper, if their efficacy could be scientifically validated.

Materials and Methods

Selection of the plants

The plants were selected mainly on the basis of
frequency of their being mentioned by the farmers. A total of twenty-seven
respondents (including farmers and herbalists) were interviewed. Twenty-one plants
were mentioned. Of these, seven were selected: Tetradenia ripara (kyewamala), Cannabis sativa (njaga), Moringa oleifera (moringa), Carica
papaya (papaali), Cassia occidentalis (mutanjoka), Momordica foetida
(bombo) and Erythrina abyssinica (ejirikiti).All the seven selected
plants were collected from the field and transported to Makerere University
Botany Department for botanical identification and voucher specimens
(Waswa/04/ASCA) have been kept at the department of Veterinary Physiological
sciences, MakerereUniversity, Kampala, Uganda.

Extraction procedures

Fresh
leaves and stems of the plants were ground separately in a mortar. Each of the
plant tissues was soaked in approximately 400ml of 95% ethanol on an electrical
shaker for three hours at room temperature and then left to stand overnight.
The mixtures were filtered into conical flasks using Whatman filter paper No.
1. The filtrate was then concentrated on a rotary
evaporator at 50oC to yield semi-solid masses whose weights were
determined. The extracts were then stored in a refrigerator at 4oC.

Collection and maintenance of
the worms (Ascaris suum)

The
worms were collected from a pig abattoir (Wambizi -, Rubaga division, Kampala).
They were got from intestines of freshly slaughtered pigs, whose contents were
manually strained until all the worms were ejected. The worms were immediately
placed in two thermos flasks containing Goodwins physiological solution at 37oC.
In the laboratory, the worms were gently rinsed in distilled water at 37oC
and then placed in two large glass jars containing Goodwins physiological
solution. This setup was left in a water bath at 37oC until 6 pm when the
experiment commenced.

Screening for ascaricidal
activity and determination of ED50 of the extracts

Seven conical flasks
of 500ml capacity were labeled with different ethanol extracts. 15ml of the
respective 1% crude ethanol
extracts (prepared by diluting stock solution with distilled water in a ratio
of 1:4) and 300ml of Goodwins physiological solution were added to each of the
flasks with one control containing only Goodwins physiological solution. Ten worms
were placed in each of the eight flasks and these were incubated at 37oC
in two water baths. A similar setup using 5% solutions was also done after
the end of the first test. The worms were monitored every 12 hours for 48 hours.
The
ED50 was the dose that killed immobilized 50% of the worms within
24
h.

Five
separate parts of 10ml solution were removed from each of the selected four
stock solutions of the plant extracts and placed in different beakers. Each 10
ml sample was diluted with an appropriate volume of distilled water to make
concentrations of 2%, 1%, 0.5%, 0.25% and 0.125%.

Five flasks were labeled and filled with 300ml of
Goodwins physiological solution and 15ml of the corresponding diluted
extracts. Ten worms were then placed in each of the flasks, with a sixth flask
as a control containing only Goodwins physiological solution. The setup was then
incubated at 37oC for 48 h. The worms were monitored at 12 hourly
intervals, which was at 7p.m. and 7 a.m. daily. This procedure was repeated for the other
three plant extracts and the results obtained were recorded.

Results

Plants used for the treatment of helminthiasis

Tetradenia riparia was the most mentioned plant (over 50%) by the respondents, followed by Erythrina
abyssinica (Table 1). The least mentioned plants included Azadirachta indica and Ricinus communis.There was a very small relationship between
plants most frequently mentioned and their efficacy/ potency. Although Tetradenia
riparia and Cassia occidentalis showed significant ascaricidal
activity, Erythrina abyssinica was only moderately ascaricidal. Carica
papaya, another less commonly mentioned plant (14.9%) showed relatively
high ascaricidal activity.

Table 1. Plants mentioned
for the treatment of helminthiasis in the study area

Plant (Luganda name in brackets)

Frequency (f)

Percentage (%)

1.

Tetradenia riparia (kyewamala)

14

51.9

2.

Erythrina abyssinica (Ejirikiti)

11

40.7

3.

Cassia occidentalis (Mutanjoka)

8

29.6

4.

Momordica foetida (Bombo)

8

29.6

5.

Euphorbia hirta (Kisandasanda)

8

29.6

6.

Vernonia amgydalina (Mululuza)

7

25.9

7.

Phytolacca dodecandra (Luwoko)

6

22.2

8.

Moringa oleifera (Moringa)

5

18.5

9

Senna didyomobotrya (Mukyula)

5

18.5

10.

Steganotaenia araliacea (Kinulangombe)

4

14.8

11.

Cannabis sativa (Njaga)

4

14.8

12

Carica papaya (Papaali)

4

14.8

13

Combretum collinum (Mukoola)

4

14.8

14.

Nicotiana tabacum (Tabba)

4

14.8

15.

Bridelia micrantha (Katazamiti)

3

11.1

16.

Vangueria apiculata (Amatuguda)

3

11.1

17

Teclea nobilis (Nzo)

3

11.1

18.

Ricinus communis (Nsogasoga)

2

7.4

19.

Justica exigua (kazunzanjuki)

2

7.4

20.

Bidens pilosa (Sere)

2

7.4

21.

Azadarichta indica (Niimu)

2

7.4

Preliminaryscreening
of the extracts for anthelmintic activity

From the initial results obtained from screening of the
seven plants, it was observed that only two plants, Tetradenia riparia and Cassia occidentalis showed activity at 1% concentration, with Tetradenia
riparia killing three and Cassia occidentalis killing two worms
respectively (Tables 2 and 3). The rest of the plants did not show any activity
at this concentration after 48 h. At 5% concentration, T. riparia and C.
occidentalis killed the worms within the first 12 h, C. papaya after
24 , M. foetida after 36 h and E. abyssinica after 48 h. C.
sativa and M. oleifera achieved only a limited effect after 48 h.
Hence Tetradenia riparia and Cassia occidentalis were potentially
the most efficacious of the selected plants, with Cannabis sativa and Moringa
oleifera on the opposite side of the scale.

Effect of various
concentrations and incubation time of Tetradenia riparia extract on Ascaris
suum

The ascaricidal
effect of T. riparia extract increased with increasing concentration of
the extract and the incubation time as shown in Table 4. The lowest
concentration of 1.25 mg/ml showed some ascaricidal activity by 12 h. and
maximum effect in 48 h. Doubling concentration achieved a similar effect 12 h
earlier while a concentration of 20mg/ml killed all worms within 12 h. Analysis
of variance revealed a significant difference in ascaricidal activity for the
different concentrations of T. riparia extract (P = 0.019) but for
different incubation periods (P = 0.067).

Table 4. Effect
of various concentrations and incubation time of Tetradenia riparia extract
on Ascaris

Conc. (mg/ml)

Total worms used

Number of worms
dead

Time (hrs) 12 24
36 48

1.25

10

2

4

7

10

2.50

10

3

7

10

10

5.00

10

6

9

10

10

10.00

10

8

10

10

10

20.00

10

10

10

10

10

Table 5. Effect
of various concentrations and incubation time of Cassia occidentalis extract
on Ascaris

Conc. (mg/ml)

Total worms used

Number of worms
dead
Time (hrs) 12 24
36 48

1.25

10

0

1

3

6

2.50

10

2

3

5

9

5.00

10

4

6

8

10

10.00

10

7

10

10

10

20.00

10

10

10

10

10

Table 6. Effect
of various concentrations and incubation time of Carica papaya extract
on Ascaris

Conc. (mg/ml)

Total worms used

Number of worms
dead
Time (hrs)

12 24
36 48

1.25

10

0

0

1

2

2.50

10

0

0

3

4

5.00

10

1

2

4

5

10.00

10

3

4

6

7

20.00

10

5

8

10

10

Table 7. Effect of various concentrations and incubation time of Momordica foetida extract
on Ascaris

Conc. (mg/ml)

Total worms used

Number of worms
dead
Time (hrs) 12
24 36 48

1.25

10

0

0

0

1

2.50

10

0

0

1

3

5.00

10

0

2

3

4

10.00

10

2

3

5

6

20.00

10

4

6

8

10

Effect of various
concentrations and incubation time of Carica papaya extract on Ascaris
suum

There was a
distinct relationship between the incubation time and concentration of Carica
papaya extract with the ascaricidal activity of the extract as shown in
Table 6. An increase in incubation time and concentration of the extract
resulted in higher mortality of the worms. Both the 1.25 mg/ml and 2.5 mg/ml concentrations
were effective in the first 24 h and neither achieved ED50sof
the end experiment. ED50s were, however, achieved by all higher
concentrations, 5 mg/ml in 48 h, 10 mg/ml in 36 h and 20 mg/ml in 12 h. Analysis
of variance also revealed a significant difference in ascaricidal activity for
both the different incubation periods of C. papaya extract (P <
0.001).

Effect of various
concentrations and incubation time of Cassia occidentalis extract on the
mortality of Ascaris

The ascaricidal
activity of C. occidentalis increased with incubation time and
concentration of the extract as shown in Table 5. While the minimum
concentration of 1.25mg/ml killed 60% of the worms after 48 h., a double
concentration of 2.5mg/ml killed 90% of the worms by the end of the experiment.
The median dose of 5.0 mg/ml took effect by 12 hours, killing 40% of the worms
and over 50% of the worms 24 hours. All worms were dead by 48 hours. A
concentration of 10.00 mg/ml killed 70% of the worms by 12 hours and all by 24
hours. The highest concentration had killed all the worms by the first
observation. Analysis of variance revealed a significant difference (P = 0.000037)
in ascaricidal activity for different concentrations of Cassia occidentalis extract. Analysis of variance also revealed a significant difference (P = 0.002)
in ascaricidal activity for different incubation periods of Cassia
occidentalis extract.

Effect of various
concentrations and incubation time on Momordica foetida extract on the
mortality of Ascaris

The ascaricidal
activity of M. foetida is shown in Table 7. 1.25mg/ml killed 10% of the
worms by 48 h; 2.5mg/ml killed 10% by 36 hours and 30% by 48 h. A dose of 5.0 mg/ml
killed 20% by 24 h. and 40% by the end of the experiment; while 10.00 mg/ml
killed 50% of the worms after 36 hours and only 10% more after 48 h. A dose of
20.00 mg/ml killed over 50% of the worms by 24 h. and 100% by 48 h. Analysis of
variance revealed a significant difference (P=0.000037) in ascaricidal activity
for different concentrations of Momordica foetida extract.

Analysis of variance also
revealed a significant difference (P=0.002) in ascaricidal activity for
different incubation periods of Momordica foetida extract.

The median effective doses (ED50s) of the
plant extracts

The median effective doses of the
plant extracts were obtained (using the computer program Microsoft Graph 2000
Chart). The ED50 of Tetradenia riparia was found to be
1.62mg/ml, that of Cassia occidentalis was 4.13mg/ml, Carica papaya had a median effective dose of 12.50mg/ml and Momordica foetida had an
ED50 of 16.75mg/ml.

Discussion

This study showed
that some plants used in ethno veterinary medicine could be of value in the
treatment of helminthiasis. Out of the seven plants studied, five yielded
appreciably positive results, four of which had ED50s lessthan20mg/ml within 12-36 h. These were T. riparia, Cassia
occidentalis, Carica papaya, Momordica foetida, and Erythrina
abyssinica. These findings agree with previous reports that indigenous plants
are useful in the treatment of helminthiasis (Akhtar and Riffat, 1984). Two of
the studied plants (Cannabis sativa and Moringa oleifera), however,
did not give satisfactory results, with ED50s more than 50mg/ml in
the initial screening.

The anthelmintic
property of plants is dependent on numerous substances that are found in them.
These could be alkaloids, sugars, saponins, aromatic oils, resins and other
medicinally useful chemicals (Lejoly et al., 1996). Oryema (1997)
reported that substances like steroids, coumarins, tannins, and triterpoids and
other chemical constituents of plants like alkaloids, glycosides, enzymes, anthraquinones,
tannins, gums, fixed oils, fats, waxes, volatile oils, proteins and
carbohydrates all have medicinal or pharmaceutical value. Many species of Cassia especially Cassia occidentalis, Cassia senna, and Cassia tora are commonly used in traditional medicine in tropical Africa for the treatment
of worm infections, constipation, pleurisy, edema, ringworm and eruptive skin
lesions (Weiser, 1994). Cassia senna contains anthracene glycosides (anthraquinone
derivatives), one of the important plant drug constituents (Wagner et al.,
1984). The milky juice of Carica papaya contains proteolytic ferments,
which together with papain have successfully been used as an anthelmintic agent
for the treatment of Ascariasis, Trichuriasis, and ancylostomiasis (Watt
and Breyer-Brandrijk, 1962).

The anthelmintic
effects of Cannabis sativa was relatively low compared with other
extracts in this study. This was probably due to the mode of action of the
active principle. Balick and Cox (1996) stated that Cannabis sativa effects are almost completely confined to the cerebral hemisphere resulting in
characteristic fibrillary tremors and ataxia due to motor in-coordination. Its
most active compound is a resin containing delta-1- tetrahydrocannabinol (THC). Erythrina abysinnica, another of the tested plants yielded fairly low
anthelmintic activity. Its seeds contain alkaloids like erythrovine, erythroline
and erysothiopine (Watt and Breyer, 1962). Momordica foetida, a
multipurpose plant, also gave moderate results. It is used as an abortifacient
and ecbolic, among others. The plant has been shown to exhibit some
antimalarial activity and the root extract contains foetidin as a major
chemical constituent ((Waako et al., 2005 and Marquis et al., 1977).

In
conclusion, therefore,this study has been able to demonstrate
significant ascaricidal activity in some plants which could be used in
ethno-veterinary medicine. It is recommended that the extracts of T. riparia,
E. abyssinica, M. foetida, C. papaya and C. occidentalis should
be further analyzed to isolate the probable anthelmintic principles in them. Toxicity
studies of the effective plants should also be done to determine the safety indices
of the extracts. Studies to determine the mechanisms of the action,
compatibility with other drugs, side effects and other important parameters
should be done.